A comprehensive study on essential oil compositions, antioxidant, anticholinesterase and antityrosinase activities of three Iranian Artemisia species

Artemisia is one of the most diverse genera in the Asteraceae family. The genus is wildly distributed in Irano-Turanian habitats and includes 34 species in Iran. Here, for the first time the essential oil variability, antioxidants and anti-cholinesterase and anti-tyrosinase activities of extracts of three Artemisia species (A. tournefortiana, A. khorassanica, A. haussknechtii), from different regions of Iran were evaluated. Based on GC–MS analyses, 81.84% to 98.70% of the total oils were identified. Cluster analysis grouped the studied populations in three different chemotypes. The highest and the lowest essential oil contents were observed in A. khorassanica and A. haussknechtii species, respectively. Camphor, en-in-dicycloether, 1,8-cineole and (Z)-β-farnesene were the dominant components of essential oil in investigated ecotypes. The results revealed that the total phenol content was higher in A. tournefortiana collected from Kerman and A. haussknechtii collected from Chaharmahal and Bakhtiari. However, the lowest phenol content was recorded for A. haussknechtii collected from Isfahan province. The highest flavonoids content was found in A. tournefortiana collected from West Azerbaijan and A. khorassanica collected from North Khorasan. The highest FRAP antioxidant activity was observed in A. tournefortiana (Kerman) and the lower amount was in A. haussknechtii collected from Kohgiluyeh and Boyer-Ahmad. The highest antioxidant activity by DPPH method was in A. khorassanica collected from South Khorasan and the lowest activity was in Isfahan's A. haussknechtii. The acetycholine esterase inhibitory activity was higher in A. tournefortiana collected from West Azerbaijan; and the lowest activity was in A. haussknechtii collected from Chaharmahal and Bakhtiari province. The highest tyrosinase inhibitory activity was in A. khorassanica collected from North Khorasan; and the lowest was in A. haussknechtii collected from Chaharmahal and Bakhtiari.

The genus Artemisia has a great distribution throughout the world and comprises 34 annual, biennial or perennial species throughout Iran 1 . Most of the Artemisia species are perennial, however about ten species are considered to be annual or biennial plants 2 . Artemisia species are mainly distributed in Asia, Europe and North America 3 . In Iran the distribution of Artemisia species is depends on various factors such as altitude and climatic conditions.
The genus Artemisia L. comprises medicinally important plants with valuable phytochemicals having a vast array of biological activities 3 . Over the years, more than 600 secondary metabolites belonging to different classes have been identified from Artemisia species, with valuable medical properties 4 . For thousands of years, the Chinese have been used A. annua as herbal tea preparation against malaria. Artemisia is considered to have a great potential for its biological activities, especially for treating viral infection and inflammation 5 . Artemisinin as the principal anti-malarial compound isolated from A. annua and the World Health Organization has recommended artemisinin-based combined therapies for the treatment of malaria 6 . Previous studies have been reported the insect's repellent and insecticidal properties of the essential oil and the healing properties of A. khorassanica extract 7,8 . Recently, studies show that Artemisia amygdalina protects neurons through upregulation of Nrf2 pathway and may have the possibility to be a therapeutic agent for Alzheimer disease 9 . Ethanolic extract of Artemisia haussknechtii has antibacterial properties 10 .
Essential oils are among the important secondary metabolites of medicinal and aromatic plants. They have many usages in various industries and fields; from the pharmaceutical and cosmetic to the food and aromatherapy industries. Because of the various essential oil components, especially in leaves and flowers, some species of Artemisia genus possess a strong aroma. Many studies have shown that Artemisia species display significant intraspecific variations in the essential oil constituents. Various factors are involved in the diversity of essential oil compounds; such as pH, climatic factors and etc. In some cases, the variation in the volatile components of these plants may occur during plant ontogeny or growth at different altitudes 3 .
These days, researchers are paying attention to herbal medicines because of their fewer side effects; and there are some studies about medicinal plants inhibitory effects on acetylcholine esterase. For example, in a study concluded by Shekarchi et al., relatively non-polar components of F. persica var. persica had AChEI activity 11 ; or methanolic extract of Mentha pulegium had significant effect on the activity of this enzyme 12 . Acetylcholine is the neurotransmitter at synapses and within the central nervous system 13 . Alzheimer's disease (AD) is one of the most common forms of dementia. The reduction in acetylcholine synthesis is the main cause of AD. Therefore, increasing the cholinergic levels in the brain by inhibiting the biological activity of acetylcholinesterase (AChE) is one of the potential therapeutic strategies for preventing AD 14 .
Tyrosinase is an enzyme that is widely distributed in different organisms of plants and has an important role in the melanogenesis and enzymatic browning. Browning of fruits, fungi and vegetables is a common undesirable phenomenon. Tyrosinase is the main enzyme responsible for this enzymatic browning. Therefore, its inhibitors are attractive in medicinal industries as depigmentation agents and also as anti-browning compounds in food and agriculture industries 15 .
There is no any comprehensive research on three species of A.tournefortiana, A.khorassanica, and A.haussknechtii, so this research was designed to evaluate the variation in essential oil composition of these species as well as TFC, TPC, antioxidants, anti-cholinesterase and anti-tyrosinase activities of extracts.

Materials and methods
Reagents and standards. Folin and Ciocalteu's phenol reagent, 2,2-Diphenyl-1-picrylhydrazyl Free Radical (DPPH), gallic acid, tannic acid, and n-alkanes were purchased from Sigma-Aldrich company (MO, USA). AlCl3, HCl, NaHCO3, HPLC grade methanol and GC grade n-hexane were purchased from Merck company (Darmstadt, Germany). Other chemicals and solvents were analytical grade and were purchased from Merck (Darmstadt, Germany). Plant materials, extraction and analysis of essential oil. The Artemisia species from Iranian provinces were collected and identified by the laboratory staff of Iranian Biological Resource Center (IBRC). All accessions were obtained under national and international guidelines and the plants were collected under the supervision and permission of Tabriz University and all authors comply with all the local and national guidelines.
Aerial  Figure 1 shows a schematic diagram of the experiments.
Fifty grams of air-dried powdered plants (aerial parts) were subjected to hydro-distillation using a Clevenger type apparatus for 3-4 h (until the essential oil volume remained constant). Water to plant material ratio in hydro-distillation process was 10:1, the rate of distillation was about two drops per second.
Determination of total phenolic contents. Methanolic extracts (1:10) (methanol 80%) were used for total phenolic assay. For the extraction procedure, 1 g of powdered aerial parts samples were dissolved in 10 mL methanol: water 80:20 mixtures. The procedure was continued by shaking the samples for 72 h. After centrifugation, the supernatants were used for the assays. The method of Stankovic (2011) 16 and Velioglu et al. (1998) 17 with a slight change was applied to measure total phenolic content, using the folin-ciocalteu regent. To obtain the calibration curve 5 ml of folin-ciocalteu regent was dissolved in 50 cc distilled water and prepared the sodium bicarbonate solution (7.5%). So, different concentrations of gallic acid standards (100, 50, 25, 12.5 mg/L) by dissolving gallic acid in methanol 80%. Then, 3 ml of folin-ciocalteu solution and 3 ml of sodium bicarbonate solution were poured into the 15 mL centrifuge tubes and 100 µl of the extracts were added to this mixture. These solutions and standards were placed in water bath (45°) for 30 min. The control sample was mixture of 10% folinciocalteu and sodium bicarbonate7.5%. Finally, the absorbance was read at 765 nm with a spectrophotometer (Camspec-Model M550).

Determination of total flavonoid contents.
To measure the amount of total flavonoid, aluminum chloride )2%( was dissolved in methanol (80% in water). Then, we poured 1 ml of this solution and 1 ml of extract into the 15 mL centrifuge tubes, and incubated them at room temperature for 1 h. Quercetin standard solutions were made. At the end, the absorbance was read at 415 nm with spectrophotometer (Camspec-Model M550). The blank sample was aluminum chloride 2% solution 16 .
Estimation of total antioxidant activity. DPPH method. DPPH method was used to measure the antioxidant content of the extracts. Briefly, 10 mg of DPPH was dissolved in 25 ml of methanol. Different concentration of the extracts (10, 20, 50, 100 μL) were prepared. 100 µl of sample (different concentrations) was dissolved in 200 µl of DPPH solution (0.2 mM); and then, the volume was increased to 400 µl with 80% methanol. After keeping in the darkness at room temperature for 30 min, the absorbance at 517 nm was investigated. Methanol and ascorbic acid were used as control and positive control samples, respectively. Ascorbic acid concentrations were prepared below 100 mg/kg (6.25, 12.5, 25, 50, and 100 mg/kg).
FRAP method. Benzie and Strain (1996) 18 method was used to evaluate the antioxidant activity with some changes. Briefly, 300 mMol acetate buffer was prepared with pH 3.6 (3.1 gr sodium acetate was dissolved in 16 mL and the volume was increased to 1 L with distilled water), then 0.062 gr TPTZ (Tripyridyltriazine) was dissolved in 10 cc choloridric acid 40 mMol and 0.054 gr of FeCl 3 .6H 2 O was dissolved in 10 mL distilled water. To prepare FRAP solution, acetate buffer, TPTZ and iron chloride (10:1:1) were mixed together at 37 °C.
In this experiment 600μL of FRAP solution was added to 30μL methanolic extract and were kept at 37 °C for 8 min. The blank sample was FRAP solution. Fe2SO4.7H2O solution was prepared with different concentrations (500, 250, 125, 62.5 and 31.25 mg/kg) to draw a standard curve. Then, the absorbance was red at 593 nm with spectrophotometer (Camspec-Model M550).

Results and discussion
Essential oils. The essential oil content of different ecotypes was between 0.02 and 0.1 ml (A.haussknechtii; A.khorassanica). In general, the average yield of essential oil was higher in A. khorassanica species and lower in A. haussknechtii species. Different ecotypes of A. haussknechtii species did not noticeably differ in essential oil yield. Few studies have been done on the composition of essential oils of these three species (A. khorassanica, A. haussknechtii, A. tournefortiana) in Iran. The major constituents of A. khorassanica essential oils were reported as 1,8-cineol, camphor, and davanone 22 . In another study, α-thujone, β-thujone and camphor were the main constituents of A. khorassanica essential oils 23 . Hadian et al. (2013) 24 found that oxidated sesquiterpenes are the most essential oil constituents of the aerial parts of A. khorassanica during the flowering stage. Davanone, p-cymene, Z-citral, β-ascaridol and thymol were identified as the main components of A. khorasanica essential oil. The plants were collected from south of Khorasan (Saride village). In A. tournefortina (collected from India, Kashmir), cis-spiroether, Z-β-farnesene, trans-nerolidol and camphor were found to be the major constituents 25 .
The main components of A. tournefortiana essence, collected from Firuzkuh (Iran), were E-thujone, sabinene and β-pinene 26 . In A. haussknechtii collected from Kermanshah, camphor, α-Terpineol, davana ether, and bornyl acetate were the major components 10 . Another study shows that the main components of the volatile oil of this species collected from north-west of Iran were 1,8-cineol, camphor, artemisia ketone, fragranol, yomogi alcohol and β-pinene 27 , which strongly supports the findings of our research. Essential oils compounds were analyzed using GC-MS. According to the analysis of chromatograms, the essential oil compositions of Artemisia ecotypes are listed in Table 1. 81.84% of total oil for A.tournefortiana that collected from Kerman were identified. (E)-nerolidol (13.03%), (Z)-nerolidol (8.08%), camphor (7.69%) and (Z)-β-farnesene (7.65%) were the major components. 43.13% The identified compounds were sesquiterpenes and 34.90% of essential oil compounds were monoterpenes (Table 1).
Almost all the compounds (98.70%) in A.khorassanica essential oil collected from North Khorasan were identified. The main compounds of oil were camphor (74.22%) and 1,8-cineole (22.91%). 98.06% of the identified essential oil compounds were monoterpenes and other compounds were non-terpenoid (Table 1).
The major compounds between the essential oils of ecotypes were en-in-dicycloether, camphor, 1,8-cineole, and (Z)-β-farnesene (Table 1). En-in-dicycloether have shown important insecticidal and acaricidal effects 28 . Camphor has been used in traditional medicine over centuries, probably most commonly as a decongestant. It is used as a topical medication as a skin cream or ointment to relieve itching from insect bites, minor skin irritation, or joint pain. Said to β-farnesene possess DPPH free radical scavenging, anticarcinogenic, antibacterial, and antifungal activity and it also had demonstrated dose-related neuroprotective effects on cultured rat primary cortical neurons, blocking H 2 O 2 -induced intracellular LDH release and reduced DNA damage 47.8%, suggesting application in neurodegenerative diseases 29 .
Based on cluster analysis of essential oils compounds the investigated populations were divided into three different clusters. Isfahan's (A.haussknechtii) and South Khorasan's (A. khorassanica) populations had the highest amounts of en-in-dicycloether (47-83%) and 1-decyl-Cyclopentanecarboxylic acid (6.1-8.2%) and placed in a separate cluster. Interestingly these components have been identified for the first time as the main compounds of the species. North Khorasan's population (A.Khorassanica) with highest amount of camphor placed in a distinct cluster. Previously, camphor has been reported as one of the major components in A. khorassanica, which was collected from the north of Iran and Khorasan 22,23 . The rest of the populations placed in a separate cluster (Fig. 2).

TPC.
A. tournefortiana which collected from Kerman with 1.35 ± 0.01 mg g −1 DW and A. haussknechtii collected from Chaharmahal and Bakhtiari province with 1.34 ± 0.005 mg g −1 DW had the highest amount of total phenol content compared with other regions. After them, Artemisia of West Azerbaijan region (A. tournefortiana) with 1.30 ± 0.0 mg g −1 DW had more total phenol. Then the highest amount of total phenol belongs to A. khorassanica collected from North Khorasan and South Khorasan and they did not differ significantly. After them, total phenol of A. tournefortiana collected from North Khorasan with 1.18 ± 0.001, Semnan's A. khorassanica with 1.09 ± 0.007 and A. haussknechtii collected from Kohgiluyeh and Boyer-Ahmad province with 0.97 ± 0.003 mg g −1 DW was higher. The lowest amount of phenolic compounds belongs to Isfahan region (A. haussknechtii) with 0.92 ± 00.7 mg g −1 DW (Fig. 3). Previous study on A. biennis Willd, showed that, the hydroethanolic extract of the plant had the highest amount of phenolic content and antioxidant activity 31 . Another research on A. absinthium demonstrated that the ethanolic extract had more TPC than A. dracunculus and A.  tournefortiana with 0.97 ± 0.012 mg g −1 DW had higher total flavonoids and the Isfahan's ecotype (A. haussknechtii) with 0.93 ± 0.01 mg g −1 DW had the lowest total flavonoids (Fig. 4).  www.nature.com/scientificreports/ showed good antioxidant activity that did not differ significantly. Also, the ecotype belonging to Kohgiluyeh and Boyer-Ahmad (A. haussknechtii) with 68.13 ± 0.50 μg/mL showed the least activity (Fig. 5).  (Fig. 6). Simple correlation analysis showed a significant relationship between the TFC and TPC content (r = 0.61, p < 0.05). Furthermore, correlation analysis revealed that 1,8-cineole content is correlated with camphor (r = 0.86, p < 0.01). Moreover, there were negative correlations between en-in-dicycloether and TFC content  www.nature.com/scientificreports/ (r = -0.67, p-value < 0.05). However, there was no any significant correlation between antioxidant activity and essential oil components (Table 3).

Antioxidant activty (FRAP
Acetyl cholinesterase inhibitory activity. According to  35 . Another study found that microwave assisted extraction of A. pallens had a higher tyrosinase inhibitory effect than soxhlet extraction 36 .

Conclusions
There were variations in main components of essential oil among species and ecotypes. These variations are probably related with different environmental conditions of the plants. So, due to the various compounds in essential oils, different ecotypes can be used in different industries. Among the ecotypes of A. tournefortiana, the ecotype belonging to Kerman had the highest essence yield and the ecotype of North Khorasan had the lowest yield. Among the ecotypes of A. khorassanica species, the ecotypes belonging to the North and South Khorasan had the highest and the lowest essential oil yields, respectively. There was not much difference between the essential oil yields of A. haussknechtii ecotypes, but essence yield of Kohgiluyeh and Boyer-Ahmad ecotype was slightly lower than the others. Isfahan's A. haussknechtii methanolic extract had the lowest TFC, TPC, and antioxidant activity with FRAP method. The lowest level of antioxidant activity with DPPH method, was in A. haussknechtii collected from Kohgiluyeh and Boyer-Ahmad. Kerman's A. tournefortiana methanolic extract had the most TPC and antioxidant activity by DPPH method. A. haussknechtii collected from Chaharmahal and Bakhtiari, similar to the Kerman's A. tournefortiana had the most TPC, but the latter ecotype had the lowest tyrosinase and acetyl cholinesterase inhibition. A. khorassanica collected from North Khorasan had the most TFC and the highest tyrosinase inhibition, but the one collected from South Khorasan had the most antioxidant activity with FRAP method. West Azerbaijan's A. tournefortiana similar to the North Khorasan's A. khorassanica had the most TFC and the highest acetyl cholinesterase inhibition. Thus, these two species are the superior species with the best medicinal value, which can be introduced for cultivation in different regions of Iran and other regional countries.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
Received: 13 February 2022; Accepted: 22 April 2022 Table 3. Correlation coefficients among antioxidants and major essential oil components of Artemisia accessions. *Correlation is significant at the 0.05 level. **Correlation is significant at the 0.01 level. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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